Integrated epr nmr with frequency agile gyrotron

ABSTRACT

A frequency agile gyrotron for use in combination with an NMR system is disclosed. The frequency agile gyrotron combined with EPR-NMR magic angle spinning resonators and cryogenic sample cooling may increase the sensitivity of solid state NMR with DNP.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/310,509, filed on Nov. 11, 2016, which is herebyincorporated by reference herein in its entirety. U.S. patentapplication Ser. No. 15/310,509 is a U.S. National Phase Application ofWO Application No. PCT/US2015/030333, filed Nov. 11, 2016, which ishereby incorporated by reference herein in its entirety. WO ApplicationNo. PCT/US2015/030333 claims the benefit of priority to U.S. ProvisionalApplication No. 61/993,595, filed on May 15, 2014, which is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to frequency agile gyrotrons used toimprove magnetic resonance experiments.

BACKGROUND

Dynamic Nuclear Polarization (DNP) has emerged as a powerful strategy toincrease the sensitivity of NMR experiments on a wide range ofbiological systems by transferring the large polarization of electronspins (EPR) to nuclear spins (NMR). Crucial to the successfulimplementation of DNP in conjunction with magic angle spinning (MAS) hasbeen the development of gyrotrons and NMR probes, instrumentation usedto perform DNP. DNP currently enhances the sensitivity of NMRexperiments on membrane proteins by a factor of about 50 and on modelsystems up to about 120 at 9 Tesla.

The larger gyromagnetic ratio of electron spins compared to protonspins, lower temperatures, and faster recycle delays all combine topotentially increase the NMR sensitivity by a factor of 360,000. Theassociated experimental averaging time may decrease by a factor of 133billion. Transferring 100% of the polarization from the electron spinsand cooling samples to 5 K to achieve the theoretical gains poses anongoing challenge.

The microwave source (usually a gyrotron) used in contemporary DNPexperiments is left locked on the same frequency for continuous-waveoperation during the entire experiment. This is because although DNPgyrotrons have high microwave power output levels (>10 W), they have notyet been tuned on a fast timescale in existing magnetic resonanceexperiments.

Current MAS DNP technology may experience difficulty achievingsufficient control of EPR spins. Only a fraction of the 1 GHz broadnitroxide lineshape can be covered with a non-tunable 1 MHz γB1microwave field of about 200 GHz that exerts control over the EPR spins.Others have not been able to use EPR spin labels on peptides for DNPbecause of extensive paramagnetic broadening. Therefore, there is a needfor a frequency agile gyrotron microwave source that can output shortpulses to not only sweep-through the EPR linewidth, but also to controlall of the EPR spins simultaneously with a broad excitation bandwidth.At the same time, there is a need to increase the γB1 microwave fieldstrength by about 3 orders of magnitude (from about 1 MHz to about 1GHz).

SUMMARY

In various aspects of the disclosure, a frequency agile gyrotron systemfor DNP NMR is provided that includes: an NMR spectrometer; a signalprocessor operatively connected to the NMR spectrometer; and a frequencyagile gyrotron operatively coupled to the NMR spectrometer and to thesignal processor. The signal processor receives one or more voltagesfrom the NMR spectrometer and produces a control signal. The frequencyagile gyrotron is configured to emit a broad-banded microwave outputthat includes a gyrotron bandwidth. The NMR spectrometer controls afrequency of the broad-banded microwave output via the control signal.The frequency agile gyrotron responds to the control signal on atimescale of microseconds. The gyrotron bandwidth is wider than an EPRlinewidth and an NMR frequency.

The bandwidth of the frequency agile gyrotron may be between about 10MHz and about 1000 MHz. The NMR spectrometer may further include amagnetron injection gun that includes a cathode and an anode. The one ormore voltages from the NMR spectrometer are chosen from at least one of:a cathode voltage, an anode voltage, and an acceleration voltagecomprising a voltage difference between the cathode voltage and theanode voltage. The frequency agile gyrotron may be operated as abackward wave oscillator. The frequency agile gyrotron may produce thebroad-banded microwave output at a phase and frequency stable condition,and the broad-banded microwave output may be sliced or gated to provideat least one of: a wide instantaneous bandwidth that includes shortpulses on a nanosecond scale and an adjustable power transmission lengthfor phase control. The NMR spectrometer may further include a combinedEPR-NMR magic angle spinning resonator. The system may further include ahelium cooling system for cooling a sample to below about 5 to about 60Kelvin with helium using a spinning MAS rotor as a centrifugal gascompressor.

In another aspect, a method of DNP NMR using a frequency agile gyrotronsystem that includes an NMR spectrometer operatively coupled to afrequency agile gyrotron is provided. The method includes controlling anoutput frequency of a broad-banded microwave output produced by thefrequency agile gyrotron by changing an operational voltage of thefrequency agile gyrotron in response to a control signal correspondingto at least one voltage received from a magnetron injection gun of theNMR spectrometer. The at least one voltage may be chosen from: a cathodevoltage, an anode voltage, and an acceleration voltage that is a voltagedifference between the cathode voltage and the anode voltage.Controlling the output frequency of the broad-banded microwave outputproduced by the frequency agile gyrotron may include at least one of:sweeping the output frequency on a timescale ranging from nanoseconds tomicroseconds; producing the broad-banded microwave output in shortpulses; and producing the broad-banded microwave output in a phase andfrequency stable form and gating the broad-banded microwave output withat least one nanosecond scale switches. The method may further includeat least one of: performing at least one time-domain DNP transfer;transferring polarization from electrons to a nucleus using hyperfinecouplings of greater than 10 KHz; decoupling an electron spin from anuclear spin; and manipulating EPR spins during magic angle spinning NMRand EPR experiments to measure EPR to NMR distances and orientations.The operational voltage of the frequency agile gyrotron may be changedon a timescale ranging from nanoseconds to microseconds to perform theat least one time-domain DNP transfer. The at least one time-domain DNPtransfer may be accomplished using at least one transfer mechanismchosen from: integrated solid effect, a nuclear orientation via electronspin locking, and an electron nuclear cross polarization. The method mayfurther include cooling a sample to below about 5 to about 60 Kelvinwith helium using a spinning MAS rotor as a centrifugal gas compressor.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures illustrate various aspects of the disclosure.

FIG. 1 is an illustration of a 197 GHz high-power gyrotron oscillatorfor frequency-agile DNP.

FIG. 2A is projected nitroxide and BDPA EPR lineshapes and DNPenhancement profiles overlaid on projected power vs. frequency plot(from a 250 GHz gyrotron). Decoupling frequencies are marked as ω_(DEC)and enhancement frequencies are marked as ω_(DNP). FIG. 2B shows pulsesequence schemes achievable with a frequency agile gyrotron.Electron-nuclear decoupling and frequency modulation of the gyrotronoutput frequency will enable improvements in DNP performance.

FIG. 3 is a pulse sequence for electron dephased REDOR.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, and 4G are schematic representations ofNMR DNP probe instrumentation.

FIGS. 5A, 5B, 5C, 5D, 5E, and 5F are modulation schemes of the anodevoltage accomplished with the circuit shown in FIG. 6. FIG. 5A is astepped voltage and frequency switching scheme. FIG. 5B is a stepped andsinusoidal modulation scheme superimposed. FIG. 5C is a saw-toothedfunction scheme. FIG. 5D is a sinusoidal modulation from an alternating(AC) current radio frequency circuit scheme. FIG. 5E is a tangentialenvelope for adiabatic passage scheme. FIG. 5F is a stochastic voltageand frequency modulation scheme.

FIG. 6 is a scheme of a frequency control circuit of a gyrotron using anintegrated NMR EPR spectrometer.

FIG. 7 is a simulation of a microwave electromagnetic field of the DNPsample chamber.

FIG. 8A is a projected nitroxide EPR lineshape and DNP enhancementprofile overlaid on a projected power spectrum of the tunable gyrotron.FIG. 8B shows a scheme using frequency modulated MW power (marked DNP)to transfer EPR polarization efficiently to nuclei followed by adiabaticEPR inversions to measure long-range electron-nuclear distances andhyperfine decoupling during NMR signal acquisition. FIG. 8C is a graphof the chaotic operation of the gyrotron used for hyperfine decoupling.FIG. 8D shows the expansion of the highlighted time points in FIG. 8Cshowing instantaneous microwave bandwidth is much wider than thenitroxide EPR linewidth.

FIG. 9A is a graph of strong hyperfine couplings that may enablelong-range distance measurements (top) compared to weak ¹³C-¹³Ccouplings (bottom). FIG. 9B shows the dephasing of rotationalHahn-echoes with rotor synchronized adiabatic EPR inversions that mayyield curves that can be fit to precise long-range distances. FIG. 9Cshows similar curves can be fit to yield precise ¹³C-¹³C distances, butonly out to about 5 Å.

FIGS. 10A, 10B, and 10C are illustrations of a Fabry-Perot EPR resonatorfor magic angle spinning DNP.

FIG. 11 is a schematic of a miniaturization of helium recirculation forcryogenic MAS DNP that may reduce the cost and footprint of recyclinghelium and permit long-term operation at cryogenic temperatures of lessthan about 27 K.

FIGS. 12A and 12B are illustrations of a miniature MAS rotor-drivenhelium recirculation system.

FIGS. 13A, 13B, and 13C illustrate exogenous (FIGS. 13A and 13B) andmounted EPR spins (FIG. 13C). FIG. 13A illustrates TOTAPOL. FIG. 13Billustrates water soluble BDPA. FIG. 13C illustrates nitroxide spinlabels, TAOC and MTSSL (left) and chelated gadolinium (right).

Corresponding reference characters and labels indicate correspondingelements among the views of the drawings. The headings used in thefigures should not be interpreted to limit the scope of the claims.

DETAILED DESCRIPTION

NMR spectroscopy currently does not utilize higher dimension spectra toyield better resolution primarily because of the sensitivity required torecord each additional dimension. A frequency agile gyrotron may providethe sensitivity and instrumentation to overcome these limitations.

A frequency agile gyrotron (or a backward wave oscillator, BWO)microwave source that can output short pulses may allow not only tosweep-through the EPR linewidth, but also to control all of the EPRspins simultaneously with a broad excitation bandwidth. At the sametime, the γB1 microwave field strength may be increased by 3 orders ofmagnitude (from about 1 MHz to about 1 GHz). Higher power output fromthe frequency agile gyrotrons and an EPR resonator with a quality factorof about 100 will yield an about 1 GHz γB1 and enhanced control of the 1GHz broad nitroxide EPR resonance. To take advantage of the new EPRcontrol, cryogenic operation may be required to extend electron spincoherence lifetimes. In an aspect, the sample may be cooled to about 27K. The system may include a miniature closed loop helium coolingapparatus that uses the spinning sample rotor as a centrifugal heliumcompressor. Cryogenic and THz technology may be able to utilize mountedEPR spin labels to simultaneously measure multiple long-range (50±0.2 Å)electron nuclear distances.

Gyrotron oscillators, or backward wave oscillators, can have asufficient frequency and phase stability to provide a stable microwavebeam. The beam can then be sliced and manipulated with alreadyestablished semiconductor light activated switches to yield nanosecondscale pulses (GHz scale bandwidth), and also phase control by means ofadjustable power transmission lengths.

Provided herein is a frequency agile gyrotron system for use in DNP NMRor combined EPR-NMR. The frequency agile gyrotron system may include abroadband gyrotron microwave source, combined EPR-NMR magic anglespinning resonators, and extreme cryogenic sample cooling to increasethe sensitivity of solid state Nuclear Magnetic Resonance (NMR)experiments by a factor of 20,000 with novel time-domain Dynamic NuclearPolarization (DNP). This tremendous boost in sensitivity and control ofEPR spins may result in acquiring data six orders of magnitude fasterthan conventional NMR and may permit multiple simultaneouselectron-nuclear distances measurements out to 50 Å. The applications ofthis technology development and structure determination methodology mayhave applications to proteins, molecules, and chemical architectures ofstructural interest.

A gyrotron with frequency agility may be tuned by changing the operatingvoltage. Although it is possible to change the gyrotron frequency bychanging the operating magnetic field; such a method is not amenable tofast tuning schemes due to the significant inductance of the gyrotronmagnet. The same magnetic tuning previously seen in DNP gyrotrons canalso be accomplished with voltage tuning. However, a 460 GHz gyrotronfor 700 MHz DNP experiments does not have enough power (>10 W) over theentire nitroxide EPR lineshape (˜1.8 GHz broad at 16.4 Tesla, or 700 MHz1H). Exert control over all of the electron spins with a strongmicrowave field enables enhanced control the DNP Hamiltonian andimproved DNP performance.

The frequency agile gyrotron may have the ability to change the voltageand gyrotron frequency on a timescale ranging from nanoseconds tomicroseconds, which may improve DNP and magnetic resonance spectroscopy.Electron decoupling may be used with the frequency agile gyrotron, whichis analogous to proton decoupling. In addition, the frequency agilegyrotron may enable Electron Dephased Rotational Echo Double Resonance(ED-REDOR), which is analogous to classical nuclear spin dephased REDOR.The ability to control the microwave irradiation frequency of gyrotronsduring DNP may allow significantly more control over the DNPHamiltonian. As a result, beneficial interactions may be turned on anddetrimental ones turned off, resulting in significantly improvedperformance. DNP can routinely provide substantial sensitivity gains,but there are still tremendous opportunities for advancements.Frequency-agile gyrotrons can overcome many of the current limitationsof DNP including: 1) Poor performance at temperatures higher than 100 K;2) Inhomogenous line-broadening; 3) Inverse scaling enhancements withmagnetic field; 4) Paramagnetic broadening; 5) Failure at MASfrequencies >˜8 KHz; and 6) Disperse polarization.

Electron-Nuclear Decoupling in DNP

Dynamic nuclear polarization (DNP) may increase the sensitivity of NMRexperiments on a wide range of biological systems. The sensitivity ofDNP experiments is generated from transferring the large polarization(sensitivity) in the electron paramagnetic resonance (EPR) spinreservoir to nuclear spins. Strong hyperfine couplings yield fast andefficient electron to nuclear polarization transfer. However, nuclearspins with strong hyperfine couplings suffer from extensive paramagneticbroadening. The method of DNP NMR with a frequency agile gyrotron mayfirst utilize strong hyperfine couplings to transfer polarization, andthen switch on a strong electron-decoupling field. The pulse sequence inFIG. 8B implements a DNP polarization (ωDNP) period followed byhyperfine decoupling (ωDecouple). This allows close-in nuclei to quicklybecome polarized, followed by a period of hyperfine decoupling thatpermits spectroscopy on the close-in ¹³C spins on a protein and anybound ligands.

Electron spins on stable organic radicals interact with the magneticfield 657 times stronger than ¹H nuclear spins, resulting in atheoretical maximum gain in sensitivity of a factor of 657 asillustrated in the equation of polarization (Eqn. (I)) below. Thedecreasing sensitivity of NMR experiments on biomedically relevantpreparations may be compensated for with drastic gains in sensitivityprovided by transferring polarization from electron to nuclear spins(DNP), cooling samples to about 27 Kelvin, and repeating experimentsfaster by utilizing the short relaxation time of electron spins.

In biomedically relevant NMR samples, the electron to nuclear DNPsensitivity transfer works efficiently only at temperatures below about100 K. Such cryogenic temperatures also inherently boost NMR sensitivityby increasing the population of spins occupying the lower energylevel—note that temperature is a denominator in Eqn. (I). These twoenhancement effects are multiplicative, meaning the experimentallyrealistic gain in NMR sensitivity for DNP experiments performed at 27Kelvin is a factor of 5000 (or 2.5×10⁷ in time). Yet another advantageto the use of DNP in NMR experiments is that the recycle delay betweenNMR scans is governed by the relaxation properties of the electronspins, which is much faster than nuclear spins, and can result in 100times faster experimental averaging.

In an aspect, DNP may enhance the sensitivity of NMR experiments onmembrane proteins by a factor of about 50. Electron nuclear decouplingexperiments employed with a frequency agile gyrotron (see FIG. 1) mayimprove DNP enhancement factors to 500, while also reducing the recycledelays from about 3 seconds to 300 milliseconds. Advanced helium cooledNMR probe instrumentation may permit NMR experiments at temperatures aslow as about 27 K, resulting in a sensitivity gain of about a factor of20,000 and a data collection rate about 400 million times faster thanconventional solid state NMR.

The successful implementation of DNP in conjunction with magic anglespinning (MAS) for biomolecular structure determination has been enabledby the development of gyrotrons and NMR probes. However, gyrotrons thatcan switch the microwave frequency quickly have not yet been employed inDNP experiments. By switching the gyrotron frequency from 197.0 GHz to197.3 GHz on a timescale of microseconds, the EPR spins may beirradiated and partially average out the electron-nuclear dipolarinteractions with an about 2 MHz continuous microwave decoupling field(FIG. 2A). This frequency jump may be achieved by decreasing theoperating voltage of the gyrotron by about 670 V. Decoupling theelectron spins during the NMR acquisition (FIG. 2B, scheme 1) mayimprove sensitivity, since many NMR signals are extinguished due todirect interactions with the EPR spins (paramagnetic relaxationeffects). Decoupling the electron spins may further have an impact onthe NMR spectroscopy that can be performed on nuclear spins thatotherwise suffer from extensive paramagnetic broadening.

The strong electron-nuclear dipolar interaction not only broadens NMRspectra, but also creates a so-called spin diffusion barrier. Thisbarrier to nuclear polarization dispersion exists because strongelectronuclear dipolar couplings shift the resonances of the protonsclose into the polarizing agent too far in frequency from resonancesfrom “bulk” protons. The spin diffusion barrier is detrimental to DNPperformance for two reasons. The close-in protons actually drain thepolarization from the electron, hindering that polarization from gettingto the bulk spins. In addition, the very strong electron-nuclear dipolarcouplings cannot be leveraged for DNP. The couplings of up to 7 MHzyield fast and efficient DNP transfers of polarization from theelectron.

Those strong couplings may be utilized and in turn permit DNP atphysiological temperatures and higher spinning frequencies, and alsoimprove DNP enhancements at cryogenic temperatures. The pulse sequencein FIG. 2B, scheme 2, implements switched polarization (ωDNP) anddecoupling (ωDEC) periods. This enables close-in protons to quicklybecome polarized, followed by a time when the ωDEC field collapses thespin diffusion barrier to allow the polarization to be efficientlyspread to the bulk. Electron-nuclear decoupling and frequency modulationof the gyrotron output frequency enables drastic improvements in DNPperformance.

DNP experiments on membrane proteins have previously used exogenousbiradical EPR polarizing agents. Due to the about 100 Å physicalseparation between these EPR spins to the nuclear spins of structuralinterest, the enhanced EPR polarization must undergo an inefficientrelayed polarization transfer. In an aspect, the ¹³C spins may bepolarized directly with rigid amide nitroxide residues incorporated intothe protein domains.

In an aspect, similar to the orientation of the biradicals in exogenousEPR polarizing agents, rigid peptide amide nitroxides must have anorthogonal orientation of the two g-tensors. In this aspect, the 90°orientation of the amide radicals in FIG. 9C may yield a separation ofEPR frequencies that match the ¹³C Larmor frequency (75 MHz) and thuspolarize nuclear spins effectively. Solid phase peptide synthesis mayallow incorporation of nitroxide residues and selective labeling of ¹³Csites that may be important to ligand binding.

Typically, resolution is compromised due to uniform ¹³C labeling andinhomogenous broadening of cryogenic MAS experiments. In an aspect, themethod may not require uniform ¹³C labeling. Isotope labels may only beused on sites that encode important structural information on ligandbinding, such as but not limited to ¹³C on Trp252, Leu251, Met239,bryostatin and prostratin. In FIG. 9A, many of the 27 predictedcorrelation peaks may be resolved in the ¹³C-¹³C 2D NMR spectrum. Thelinewidth (1 ppm) of the predicted resonances in FIG. 9A comes from MASDNP spectra found in FIG. 8A. ¹³C-¹³C correlations peaks are ofteninhomogenously broadened in DNP spectra due to the cryogenic trapping ofmultiple conformations. However, each distinct conformation ischaracterized by correlated chemical shifts that can be exploited toincrease spectral resolution with a double quantum correlation in theindirect ¹³C dimension. Such line-narrowing strategies have not yet beenemployed to structure determination efforts of membrane proteins withMAS DNP.

Time Domain DNP Transfers with Frequency Swept or Broadband GyrotronOscillators

A phenomenon referred to as the Cross Effect is active when the EPRlineshape is wider than the nuclear Zeeman frequency. This is the casefor nitroxide radicals. For example the about 1000 MHz lineshape of thenitroxide EPR spectrum shown in FIG. 2A is greater than thecorresponding 300 MHz proton frequency. The Cross Effect can beunderstood in a cross-relaxation framework. When the microwave frequencyis targeted on the low-frequency side (about 197.3 GHz in FIG. 2A), themicrowaves burn a hole in the mostly inhomogenously broadened EPRspectrum. In other words, the Zeeman spin states of the electron spinsnear the irradiation frequency become nearly equal. When these spinsrelax back to their equilibrium Zeeman population, they cross-relaxanother electron spin on the other side of the EPR lineshape along witha nuclear spin. These nuclear spin states than become polarizedaccording to the Boltzmann distribution of the electron spin states,resulting in DNP enhancements of the NMR sensitivity.

The amount of the EPR spectrum that is saturated from the microwavefield is thus an important factor in Cross Effect DNP. If fewer electronspins are saturated, fewer spins participate in DNP and the enhancementsare smaller. It follows that a strategy that increases the saturationbandwidth of the microwave field would lead to higher DNP enhancements.A fast (>10 KHz) frequency modulation of the gyrotron frequency, withsufficient microwave power, will accomplish this. Modulating themicrowave frequency over the lower frequency side of the EPR spectrummay (shading in FIG. 2A) result in more nuclear polarization and NMRsensitivity.

Time domain DNP transfers such as the Integrated Solid Effect (ISE),Nuclear Orientation via Electron Spin Locking (NOVEL), electron nuclearcross polarization, and other irradiations schemes have been proven toyield fast, efficient transfers at low (˜9 GHz) microwave frequencies.All of these techniques could be extended to operate at higherfrequencies (100-1000 GHz) with the use of frequency swept gyrotrons (orBWOs), or frequency and phase stable gyrotrons (or BWOs) that supply amicrowave beam that can be sliced and manipulated with light activatedsemiconductors switches. All of these time domain schemes have thepossibility of transferring polarization from electrons to nuclei fastenough to allow Optical Polarized DNP at high magnetic fields, and toperform EPR to NMR polarization transfers efficiently attemperatures >200 Kelvin.

Simultaneous EPR-NMR Distance Measurements up to 50 Å

A 1/r³ distance dependence of the dipolar interaction encodesbiomolecular structure (see FIG. 9A). Although homonuclear dipolarcouplings (labelled ¹³C-¹³C in FIG. 9A) can be determined precisely tomeasure short-range distances, longer distances are more challenging tomeasure due to the weak nuclear-nuclear dipolar interaction. Theelectron-nuclear (hyperfine) interaction is 2600 times stronger due tothe large magnetic moment of the electron spin. Strong hyperfinecouplings may be used to measure electron-nuclear distances on a proteinout to about 50 Å.

FIG. 8A illustrates the projected nitroxide EPR lineshape and DNPenhancement profiles overlaid on the projected power spectrum of thefrequency agile gyrotron. This experiment may employs electron dephasedrotational Hahn-echoes and adiabatic inversions of the electron spinonly made possible with frequency agile gyrotron technology disclosedherein. FIG. 8B shows a scheme in which frequency modulated MW power(labeled DNP) transfers EPR polarization efficiently to nuclei, followedby adiabatic EPR inversions to measure long-range electron-nucleardistances and hyperfine decoupling during NMR acquisition.

Similar to heteronuclear distance measurements, the dephasing ofrotational Hahn-echoes may be monitored as a function of the EPRadiabatic inversion placement in the MAS rotor cycle. FIG. 9A showsstrong hyperfine couplings may enable long-range distance measurements(see top graph) compared to weak ¹³C-¹³C couplings (see bottom graph).FIG. 9B is a curve fit to precise long-range distances yielded by thedephasing of rotational Hahn-echoes with rotor synchronized adiabaticEPR inversions as disclosed herein. As seen in FIG. 9C, similar curvesmay be fit to yield precise ¹³C-¹³C distances, but only out to about 5Å. Due to the strength of the hyperfine interaction, it may be possibleto measure the 15-20 Å distances as indicated in FIG. 9A. In an aspect,long-range distances may be measured between rigid nitroxide labels and¹³C labels both on residues in binding pockets and on ligands with a±0.2 Å precision.

In an aspect, the transverse electron relaxation may be extended toenable adiabatic EPR inversions. This may be accomplished withdeuteration of residues near the nitroxide moiety and by cooling thesample as cold as possible. In one aspect, the sample may be cooled to atemperature less than about 27 Kelvin.

Microwave Frequency Modulation for Broad-banded Electron-NuclearDecoupling

Extending the decoupling strategies discussed herein above to DNP usingnitroxide radicals and the 3-spin Cross Effect mechanism may require afrequency modulation of the microwaves across the entire broad EPRlineshape. Such modulation of the microwave frequency from about 197.0to 198.3 GHz (see shading in top of FIG. 2A) may be accomplished bymodulating the operating voltage of the gyrotron by ±1.4 kV. Continuouswaveforms applied in NMR may have similar analogies to EPR and DNPtransitions. Analogous to the first heteronuclear decouplingexperiments, a random modulation of MW frequency over the lineshapemight better average out electron-nuclear couplings.

Magic Angle Spinning (MAS) Solid State NMR

The NMR Hamiltonian contains anisotropic terms such as dipolarinteractions and chemical shift anisotropy that can lead to shortrelaxation times and line broadening in NMR spectra of solid statesamples. However, a factor of (3 cos²θ−1) in these Hamiltonians allowseffectively averaging weaker anisotropic interactions to zero (3cos²54.7°−1=0) with mechanical rotation of the sample at 54.7° (themagic angle) with respect to the magnetic field (FIG. 7A), resulting innarrow NMR resonances.

Electron Dephased Rotational Echo DOuble Resonance (EDREDOR)

Rotational Echo DOuble Resonance (REDOR), correlates the amount ofdephasing during a spin-echo to distances between nuclear spin pairs—thecloser the “dephasing” spin is to the “observed” spin, the stronger thedephasing. Similarly, spins with larger gyromagnetic ratios yield moredephasing, enabling longer distance measurements up to about 12 A for¹⁹F-¹³C spin pairs. Electron spins have magnetic moments about 660 timeslarger than ¹⁹F nuclear spins. These strong electron spins may be usedto measure electron-nuclear distances on a protein out to about 50 Å.The pulse sequence for such an Electron Dephased REDOR (ED-REDOR)experiment is shown in FIG. 3, which includes an adiabatic inversion ofthe electron spin only made possible with frequency agile gyrotrontechnology. In typical REDOR experiments, π pulses refocusmagnetization, but in the experiment illustrated in FIG. 3, an adiabaticinversion on the electron spin interferes refocusing of the spin echo.The extent to which the magnetization is dephased directly encodes theelectron-nuclear distance, and also the orientation of the dipolarvector. Similar to REDOR, ED-REDOR may also allow the measurement of theorientation of the electron-nuclear dipolar vector in addition to themeasurement of the electron-nuclear distance.

Similar experiments exist in EPR, such as ENDOR (Electron Nuclear DoubleResonance). EDREDOR is different in a few very important ways.Primarily, ED-REDOR is conducted during a MAS experiment that yieldshigh resolution NMR spectra. The disadvantage to the MAS experiments isthe lack of an EPR resonant structure—this is why frequency agilegyrotrons are so critical. Their high power levels compensate for thelack of EPR resonant structure, enabling an adiabatic inversion of theelectron spins. Also, ENDOR is EPR detected, which limits the range ofdistance measurements to about 15 Å. ED-REDOR is also similar to solidstate NMR structural measurements with paramagnetic relaxation effects.However, ED-REDOR has a 1/r³ distance dependence versus the 1/r⁶dependence of EPRs, making it possible to measure out to about 50 Årather than 15 Å.

One of the challenges to implementing ED-REDOR is extending thetransverse electron relaxation to enable the adiabatic inversion, andlongitudinal electron relaxation time to allow for the long mixing timesrequired to measure long distances. This will be accomplished by coolingthe sample to as low a temperature as possible. In one aspect, thesample may be cooled to a temperature below about 20 Kelvin.

Polarizing agents and EPR spin labeled proteins

The stable organic radicals and EPR transition metals to be used forCross Effect DNP, Solid Effect DNP, and electron-nuclear distance anddipolar orientation measurements have not previously been used forelectron-decoupling or installing radicals on proteins for use with DNPbecause high power frequency agile gyrotrons and electron nucleardecoupling are needed. TOTAPOL (FIG. 13A) is an exogenous biradicalpolarizing agent comprised of two nitroxide moieties tethered together.The broad EPR lineshape and strong electron-electron dipolar couplingenabled by the frequency agile gyrotron is combination with TOTAPOL mayyield efficient DNP with the Cross Effect. TOTAPOL is the currently themost common polarization agent used in magic angle spinning DNP, and maybe used with in vivo studies. The broadbanded microwave irradiation fromthe frequency agile gyrotron as disclosed, which may cover the entirenitroxide lineshape, may enable powerful electron nuclear decoupling anddistance measurements. In an aspect, more narrow line EPR moieties mayalso be used.

Narrow line EPR resonances like that in water-soluble BDPA (FIG. 13B)and gadolinium (FIG. 13C), are well-suited for Solid Effect DNP,especially when high electron nutation frequencies are available. TheDNP enhancements from the Solid Effect still increase linearly withrespect to microwave power, even when gB1=3 MHz. The 100 Watt powerlevels achieved by the frequency agile gyrotron as disclosed herein andEPR resonant structures may yield very high electron nutationfrequencies in the range of about 5 MHz to about 30 MHz, or even higher.

Gadolinium is well-suited for Solid Effect DNP and electron nucleardecoupling for EPR spin labels on proteins. Although gadolinium has beenused as a polarizing agent for DNP, and also been installed on proteinsto make electron-electron measurements, performing DNP on a spin labeledprotein has proven challenging. The narrow central EPR transitionlinewidth of gadolinium is dominated by isotropic zero-field splitting,which may simplify the implementation and data interpretation ofelectron nuclear distance measurements. However, the electron spinrelaxation times of gadolinium are much shorter than nitroxides. Suchfast relaxation makes it more challenging to manipulate these spins,especially to measure electron nuclear distances. Extreme samplecooling, in one aspect to temperatures of below about 15 Kelvin mayenable combined gadolinium EPR and NMR.

TOAC (FIG. 13C) is a nitroxide amino acid incorporated with solid phasepeptide synthesis (SPPS), its rigid conformation will lead to higherprecision electronuclear measurements. MTSSL (FIG. 13C) may be installedonto cysteine residues, and may be used especially on protein GB1. Asdiscussed previously, electron nuclear decoupling and collapsing thespin diffusion barrier may permit DNP at higher temperatures (about220-273 K), where exquisite resolution in solid state NMR spectra ofmicrocrystalline GB1 has been demonstrated.

Gyrotron and DNP Probe

A gyrotron, generally disclosed herein as 100 in FIG. 1, may befrequency agile and have a wide instantaneous bandwidth. The frequencyagile gyrotron 100 may include an output window 104, an interactioncavity 106, and an electron gun 108. An electron beam 102 may extendthrough the length of the frequency agile gyrotron.

The frequency agile gyrotron 100 may be operatively connected to a NMRDNP probe 400. FIG. 4A is a schematic of a probe head for 300 MHz/200GHz magic angle spinning DNP including a microwave waveguide 404 andvacuum jacketed cryogen lines 408, 410, which may be connected from thetop of the magnet. FIG. 4B is a cross section showing the large samplevolume 412 (about 250 μL) for in vivo and extremely high sensitivity invitro studies. In an aspect, the DNP NMR probe may include a quadrupleresonant NMR coil 414. FIG. 4C shows a DNP probe that includes a heliumvariable temperature line that cools samples to between about 25 Kelvinand about 27 Kelvin, a high-performance RF transmission line circuit416, and an efficient microwave transmission line. FIG. 4D illustrates avacuum jacketed Dewar 428 for 89 mm bore magnets with connection ports430 to the top of the magnet. FIG. 4E is a schematic showing the coldhelium gas flow along to rotor surface for efficient heat-exchange, thenout exhaust ports 402 that establish a heat shield. The DNP probe mayfurther include a drive cup (turbine) 418 and at least one bearing 420.FIG. 4F is an illustration of 200 GHz radiation in a DNP cavityincluding a microcoil 422 for about 1 μL samples 426. The microcoil 422may provide improved EPR performance. Cross sections of the inductivelycoupled RF coils 422 and concave GHz mirrors 424 may generate about >1MHz nutation fields on nuclear spin RF channels and >10 MHz nutation ofelectron spins resonating at about 200 GHz. FIG. 4G is a High FrequencyStructure Simulation (HFSS) at 250 GHz of the sample chamber on theprobe in FIG. 4C. FIG. 7 is a simulation of a microwave electromagneticfield of the DNP sample chamber 412/426.

FIG. 10A is a schematic of a Fabry-Perot EPR resonator 1000 for magicangle spinning DNP. The resonator 1000 may include a magic anglespinning N₂ (g) bearing 1008 and a sapphire rotor 1014. FIG. 10Aillustrates a Teflon lens 1002 that focuses the microwave power from thegyrotron 100 to the sample 1016 using a corrugated waveguide 1004 inbetween turns of the radio-frequency coil 1006. An adjustable coppermirror 1010 may excite the cavity mode. FIG. 10B illustrates thedielectric constants (impedance) of the sample 1016 and sapphire rotor1014 are matched with anti-reflective THz coatings 1012 to reduce loss.FIG. 10C illustrates the cavity mode establishes a microwave field overthe sample 1016.

The broadband microwave irradiation generated from the frequency agilegyrotron 100 may allow significantly more control over the DNPHamiltonian. Beneficial hyperfine interactions may be able to be turnedon and detrimental ones turned off to obtain significantly moresensitive and precise biomolecular structural refinement. For instance,electron decoupling may be implemented, which is analogous to protondecoupling and electron-nuclear distance measurements that are analogousto nuclear-nuclear measurements.

The electron beam 102 ejected from cathodes in the frequency agilegyrotron 100 may generate microwave power that can interact with EPRspins at about 197 GHz. In the frequency agile gyrotron 100, theelectron acceleration voltage between the cathode and anode determinesthe microwave output and may be changed quickly to permit electronnuclear decoupling (FIGS. 8A, 8B, and 8C). For instance, a frequencyjump of about 300 MHz to switch from the DNP transfer condition todirect irradiation of the EPR resonances for decoupling may be achievedby decreasing the operating voltage of the gyrotron by about 670 V.

The about 5 Kelvin helium gas flow indicated by the arrows in FIG. 12Amay effectively cool the sample 1208 while a bearing 420 fed with coldnitrogen gas (about 60 K) provides stable magic angle spinning. In anaspect, the sample 1208 may be at about 20 Kelvin. This design employshelium exhaust ports 402 (see FIG. 12B) that force the helium to be inextended contact with the NMR rotor to increase heat exchangeefficiency, and also to cool heat shields surrounding the sample 1208.The system may further include ports 1222 (see FIG. 12B) for sampleejection, MAS bearing, MAS drive, helium cooling, and 200 GHz channel.The drive-tip 1204 (see FIG. 12A) and pressurized N₂ (g) turbine powermay provide the centrifugal force to compress helium. The arrows in FIG.12A indicate helium flow and expansive cooling of the sample 1208followed by compression. The helium may flow to a miniature heatexchanger where heat is exchanged with a second closed helium loopcooled by a cryogenic refrigerator. In an aspect, the helium may flowfrom a refrigerator 1214, to a heat exchanger for re-cooling 1216, fromthe heat exchanger 1210, and return to the refrigerator for re-cooling1212. A helium propeller 1218 and a helium impeller 1220 may be used tomove the helium through the system, as illustrated in FIG. 12A.

In the frequency agile gyrotron as disclosed herein, the operationalvoltage determines the microwave output frequency. In an aspect, thevoltage-tunable gyrotron may change the frequency by about 1 GHz inabout 1 second. The frequency agile gyrotron may allow for much fastervoltage control and adiabatic inversions through the about 1 GHznitroxide lineshape in about 2 μs. In an aspect, the gyrotron mayoperate in a “chaotic” mode of operation for hyperfine decoupling. Thephysics of the interaction of the electron beam with the interactioncavity is shown in FIG. 8C. The gyrotron quickly switches between axialmodes resulting in short (<1 ns) pseudo-random pulses that are excellentfor hyperfine decoupling. In an aspect, the instantaneous bandwidth ofthe 200 picosecond pulse in FIG. 8D may be about 4 GHz and maycompletely cover the 1 GHz nitroxide lineshape.

To perform NMR experiments relevant to biomolecular structuredetermination with DNP, probe instrumentation may perform a diverse setof tasks including; control of nuclear spins with efficientmultiresonant RF circuits, control of EPR spins with GHz irradiationusing waveguide and quasioptics, and cryogenic cooling with stable about5 KHz to about 8 KHz magic angle spinning. Spinning at the magic angle(54.7°) averages anisotropic interactions in the Hamiltonian and resultsin narrow NMR resonances and resolved spectra. The high-performancequadruple resonance NMR DNP probes must retain the capability toimplement all of the homo and heteronuclear polarization transfer anddecoupling schemes that are integral to solid-state NMR spectroscopists.

RF transmission line circuits may generate about 83 KHz nutationfrequencies on multiple RF channels and may be employed for rotors up to7 mm diameter with sample volumes of up to 250 μL (FIG. 4A, 4B, 4E, 4G).Most DNP probes typically have about 30 μL sample volumes. The new DNPprobe accommodating larger rotors will enable improved sensitivity forin vivo samples.

Sometimes it may be prohibitively difficult to make such large samplevolumes of isotopically labeled protein and drugs. Therefore,inductively coupled microcoils that house sample volumes of about 1 μLmay be incorporated into the DNP probes in an aspect. In addition toachieving excellent filling factors and sensitivity with 1 μL samplevolumes, there are many additional advantages to microcoils from a RFand GHz perspective.

Challenges to achieving high quality EPR resonators in MAS includecoupling the microwave power efficiently into the sample, and addressinglosses of microwave power from the lossy sample. The system in FIG. 4Faccomplishes both of these. Concave mirrors 424 placed in the rotoritself and opposite the waveguide aperture focus the microwave powerinto the sample 426. A 1 μL sample, although lossy, is small compared tothe large size of the EPR resonant structure, leading to high qualityfactors. Together with the high power levels achieved by the frequencyagile gyrotrons disclosed herein, EPR quality factors of 5 to 80,corresponding to electron nutation frequencies of 8-32 MHz may beachieved. Such strong control of the electron spins may lead totremendous advancements in magnetic resonance methods.

Microcoils may also generate very high nutation frequencies of thenuclear spins. With the high power amplifiers already in place on thespectrometer, high efficiency RF transmission line circuits, andmicrocoils, 0.1-1 MHz nutation frequencies may be generated on ¹H, ¹⁹F,³¹P, ¹³C, ¹⁵N, ²H; all simultaneously. Not all of the NMR pulsesequences may make use of all of these channels in the same experiment.In an aspect, the ¹H channel may be used for cross polarization and ¹Hdecoupling and the ¹⁹F and ²H channels may manipulate isotopicallylabeled spins on bryostatin. In addition, the ³¹P channel may control³¹P spins on phospholipid head groups and phosphorylated tyrosineresidues in the active site of a protein. Correlations between ¹³C and¹⁵N on uniformly labeled proteins between the ¹⁹F, ²H, and ³¹P spins mayyield not only distance constraints, but also, spectral filtering toclear-up spectral congestion. For site-resolved spectra of fully labeledproteins of >400 amino acids using ¹³C and ¹⁵N, uniquely resolved spinsand advance probe technology as described herein may be utilized.Building instrumentation that can manipulate seven types of spins(including the electron spins) simultaneously represents a huge leapforward in innovation from typical solid state NMR probes that aretriple resonance.

Among the challenges to achieving high quality EPR resonators in MASexperiments of >5 Tesla include coupling the microwave power efficientlyinto the sample, and overcoming losses of microwave power in theresonator. The Fabry-Perot resonance structure 1000 shown in FIGS. 10A,10B, and 10C accomplishes both of these objectives. An adjustable Teflonlens 1002 couples the 200 GHz microwave power 1004 from the gyrotron 100efficiently into the EPR resonator 1000 through a split radio-frequencycoil 1006 design. An adjustable concave copper mirror 1010 optimizes theboundary condition to excite a strong cavity mode (FIG. 10C).Anti-reflective coatings 1012, and a sapphire sample rotor 1014 minimizeloss of power when the microwaves pass through materials with differentdielectric constants (FIG. 10B). A High Frequency Structure Simulatormay be used to calculate the propagation of the microwaves and optimizethe materials and geometry in the EPR resonator. High quality factors(Q˜100) can be achieved because the lossy sample 1016 is small comparedto the size of the EPR resonant structure 1000. There are no EPRresonators currently available for high-frequency MAS DNP; the EPRquality factor of existing instrumentation is Q˜1.

Efficient Helium Cooled Magic Angle Spinning

Enhanced sensitivity in magnetic resonance is available at cryogenictemperature due to a 1/T dependence of the spin polarization (see Eqn.(I)). Electron and nuclear spin relaxation times also increasedrastically at lower temperatures, permitting efficient transfers of theenhanced electron polarization to nuclear spins. Consequently, most MASDNP experiments are performed at 80-100 Kelvin where inexpensive N₂ (g)can be used both to spin the NMR sample and to provide cooling. Thereare some initial studies that use about 6 L/hr of liquid helium to coolsamples to about 25 K, but are encumbered by the ever-increasing highcost of helium. Current efforts to recycle the helium entail atremendous investment in infrastructure and laboratory space (see FIG.11, bottom).

The frequency agile gyrotron system may further include a miniaturehelium recirculation system shown in FIG. 11 (top) and FIGS. 12A and12B. A centrifugal compressor 1202 mounted on the rotor harnesses therotational kinetic energy afforded by the drive-tip turbine 1204 (FIG.12A) and about 8 kHz sample rotation. The helium may circulate between aminiature heat-exchanger 1226 and the sample 1208 (see FIGS. 12A and12B). The helium flowing over the rotor may be cooled to about 5 K, butthe sample may be about 25 K due to heat conduction along the rotor andmicrowave heating.

The geometry of the rotor fins and compression manifold may yield theflow pattern shown in FIG. 12. At field strengths of about 7 Tesla, aspin at about 8 KHz may yield narrow linewidths in the MAS NMR spectra.Vacuum jacketed insulation may be critical to separate the helium loopfrom the sample chamber 1208 maintained at 90 K with N₂ (g).

Previous experiments below 10 K required tens of liters of helium anhour. There has been a renewed interest in cold helium spinning insolid-state NMR the last decade, and the drive has been to make thecooling more efficient. The length of the rotor may be extended andtight disks (baffles) used to isolate the cold sample region in thecenter of the rotor. The helium gas flow indicated by the blue arrows inFIG. 4E may be used. This design employs helium exhaust ports 402 thatforce the helium to be in contact with the rotor much longer to increaseheat exchange efficiency, and also to cool the disks surrounding thesample to establish a heat shield. These improvements, in addition tousing about 100 Kelvin MAS spinning gases, may allow sample temperaturesof less than about 20 Kelvin with minimal helium consumption. In anaspect, the helium consumption may be less than about 1 L/hr.

Spectrometer Control of the Gyrotron Frequency

In tunable gyrotrons, the acceleration voltage between the cathode andanode in the magnetron injection gun dictates the microwave outputfrequency (see FIG. 9B, right). Control of the voltage (and frequency)of the gyrotron by the NMR spectrometer enables seamless integrationbetween the NMR and EPR channels in DNP experiments. The TECMAGspectrometer may have extra arbitrary waveform generators with a 10 nsstep size. Thus, implementing the voltage and frequency agility schemesshown in FIG. 6 may be accomplished from the integrated DNP spectrometerconsole. Voltage output channels from the spectrometer may be amplified(and optically isolated), added together in a biasing circuit, andconnected to the gyrotron anode. The voltage on the anode controls theelectric potential (see FIG. 6, right), thus tuning the velocity of theelectron beam and the microwave frequency output.

Protein Sample Preparation for NMR

Previously, dozens of milligrams of protein for atomic level structuralbiology was required. In an aspect, the sample size for in vitro samplesmay be decreased. With the microcoil instrumentation and DNP sensitivitydescribed previously herein, there may be excellent sensitivity withabout 1 μL samples. For example, with about 200 μg of protein (most ofthe volume is taken up by lipids), a full-length protein in eukaryoticcells may be expressed and the functional kinase may be purified withoutneed for optimizing yields at every step of the protocol. Similarly, 200μg of a protein from solid phase peptide synthesis may enableincorporation of selectively isotopically labeled residues and EPR tags.At the same time, it will be much easier to provide about 30 μg ofsample versus the 4 mg currently needed.

In vitro measurements may benefit from DNP sensitivity. For instance thesensitivity may be leveraged to determine drug and protein confirmationspresent at a minute fraction of the cryogenically trapped ensemble.Often these thermodynamically less favored states are critically themost important structures—excursions in the energy landscape that resultin drug binding, dissociation, and catalysis.

EXAMPLES Prophetic Example 1

FIG. 4B shows the chemical structure 1,3-bisdiphenylene-2-2-phenylallyl(water soluble BDPA), the exogenous stable organic radical that may be asource of the enhanced magnetic resonance polarization (sensitivity).FIG. 2A shows the electron paramagnetic (EPR) spectra of nitroxide andBDPA radicals in red. The enhancement profiles show the level ofpolarization enhancement obtained on the nuclear spins by sweeping themicrowave irradiation frequency (or NMR magnet field strength) throughthe DNP matching conditions. For the narrow line BDPA radical, the SolidEffect, a two-spin DNP mechanism is active when the microwaveirradiation frequency is 300 MHz (the proton nuclear Larmor frequency)away from the EPR resonance. Protons are polarized from the Solid Effectwhen the gyrotron is set to 197.0 GHz and the EPR resonance is at 197.3GHz.

An in vitro NMR sample will contain about 200 micrograms of isotopicallylabeled bryostatin and also PKC C1b domain, phosphatidyl serine lipids,BDPA DNP polarizing agent, and a cryoprotecting matrix of glycerol. Thesample will be loaded into a rotor for magic angle spinning (MAS). Forin vivo ligand structural determination, about 400 mg of human cells(HeLa or similar) will be treated with isotopically labeled bryostatin,spun down, and then resuspended in a cryoprotecting glycerol matrix withdissolved DNP polarizing agent, before being centrifuged into a MAS NMRrotor.

Simultaneous radio frequency irradiation resonant with ¹H, ²H, ³¹P, ¹³C,and ¹⁵N spins from a custom designed NMR radio frequency circuit mayyield sufficient control of the nuclear spins to attenuate elements inthe NMR Hamiltonian that lead to line broadening, while also permittingthe measurement to sub-angstrom precision between ¹³C, ¹⁵N, ²H isotopiclabels on bryostatin and ³¹P spins on the phospholipid head groups.

With the microcoil instrumentation and DNP sensitivity describedpreviously herein, there may be excellent sensitivity with about 1 μLsamples. For example, with about 200 μg of protein (most of the volumeis taken up by lipids), a full-length protein in eukaryotic cells may beexpressed and the functional kinase may be purified without spending alot of time trying to optimize yields at every step of the protocol.Similarly, in a cost-effective manner, 200 μg PKC C1b domain from solidphase peptide synthesis will permit incorporation of selectivelyisotopically labeled residues and EPR tags. At the same time, it may beeasier to provide about 30 μg of isotopically labeled bryostatin analogsversus the 4 mg currently needed.

In vivo NMR spectroscopy may ensure that the PKC is bound to endogenouslipids along with all of the co-factors, anchoring proteins, scaffoldproteins, and other macromolecules present in the membrane that play arole in regulation. For these in vivo experiments, large 250 μL samplevolumes may be used—it is not difficult to culture cells and spin themdown in a centrifuge to get about 200 mg quantities; the tough part isalways the purification, refolding, and reconstituting into lipids. Thein vivo spectroscopy may be extended to primary cells and determine thestructures of bryostatin and phorbol in diseased tissue.

Long-range distances will be measured between rigid nitroxide labels onthe C1b domain and ¹³C labels both on residues in the binding pocketsand on ligands with a ±0.2 Å precision.

The examples described herein are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples included hereinrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

What is claimed is:
 1. A frequency agile gyrotron system for DNP NMRcomprising: an NMR spectrometer; a signal processor operativelyconnected to the NMR spectrometer, wherein the signal processor receivesone or more voltages from the NMR spectrometer and produces a controlsignal; and a frequency agile gyrotron operatively coupled to the NMRspectrometer and to the signal processor, the frequency agile gyrotronconfigured to emit a broad-banded microwave output comprising a gyrotronbandwidth; wherein: the NMR spectrometer controls a frequency of thebroad-banded microwave output via the control signal, the frequencyagile gyrotron responds to the control signal on a timescale ofmicroseconds, and the gyrotron bandwidth is wider than an EPR linewidthand an NMR frequency.
 2. The system of claim 1, wherein the bandwidth ofthe frequency agile gyrotron is between about 10 MHz and about 1000 MHz.3. The system of claim 1, wherein: the NMR spectrometer furthercomprises a magnetron injection gun comprising a cathode and an anode;and the one or more voltages from the NMR spectrometer are chosen fromat least one of: a cathode voltage, an anode voltage, and anacceleration voltage comprising a voltage difference between the cathodevoltage and the anode voltage.
 4. The system of claim 1, wherein thefrequency agile gyrotron is operated as a backward wave oscillator. 5.The system of claim 1, wherein the frequency agile gyrotron produces thebroad-banded microwave output at a phase and frequency stable condition.6. The system of claim 5, wherein the broad-banded microwave output issliced or gated to provide at least one of: a wide instantaneousbandwidth comprising short pulses on a nanosecond scale and anadjustable power transmission length for phase control.
 7. The system ofclaim 1, wherein the NMR spectrometer further comprises a combinedEPR-NMR magic angle spinning resonator.
 8. The system of claim 1,further comprising a helium cooling system for cooling a sample to belowabout 5 to about 60 Kelvin with helium using a spinning MAS rotor as acentrifugal gas compressor.
 9. A method of DNP NMR using a frequencyagile gyrotron system comprising an NMR spectrometer operatively coupledto a frequency agile gyrotron, the method comprising controlling anoutput frequency of a broad-banded microwave output produced by thefrequency agile gyrotron by changing an operational voltage of thefrequency agile gyrotron in response to a control signal correspondingto at least one voltage received from a magnetron injection gun of theNMR spectrometer, the at least one voltage chosen from: a cathodevoltage, an anode voltage, and an acceleration voltage comprising avoltage difference between the cathode voltage and the anode voltage.10. The method of claim 9, wherein controlling the output frequency ofthe broad-banded microwave output produced by the frequency agilegyrotron comprises at least one of: sweeping the output frequency on atimescale ranging from nanoseconds to microseconds; producing thebroad-banded microwave output in short pulses; and producing thebroad-banded microwave output in a phase and frequency stable form andgating the broad-banded microwave output with at least one nanosecondscale switches.
 11. The method of claim 9, further comprising at leastone of: performing at least one time-domain DNP transfer; transferringpolarization from electrons to a nucleus using hyperfine couplings ofgreater than 10 KHz; decoupling an electron spin from a nuclear spin;and manipulating EPR spins during magic angle spinning NMR and EPRexperiments to measure EPR to NMR distances and orientations.
 12. Themethod of claim 11, wherein the operational voltage of the frequencyagile gyrotron is changed on a timescale ranging from nanoseconds tomicroseconds to perform the at least one time-domain DNP transfer. 13.The method of claim 12, wherein the at least one time-domain DNPtransfer is accomplished using at least one transfer mechanism chosenfrom: integrated solid effect, a nuclear orientation via electron spinlocking, and an electron nuclear cross polarization.
 14. The method ofclaim 9, further comprising cooling a sample to below about 5 to about60 Kelvin with helium using a spinning MAS rotor as a centrifugal gascompressor.